Apparatus, method, system for the determination of the aggregation rate of red blood cells

ABSTRACT

Systems and methods for the determination of the aggregation rate of red blood cells. More specifically, the subject technology is used to determine the aggregation rate of red blood cells, and other parameters related to these, such as viscosity, deformability, elasticity, density, in the field of in vitro medical analyses, using optical systems after or during vibration for red blood cell disruption and redistribution. Once the detected light variation stops decreasing (e.g., a minimum is reached), complete disruption is accomplished for evaluation of the blood sample.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to and claims priority from earlier filedU.S. Non-Prov. patent application Ser. No. 14/176,307 filed on Feb. 10,2014, U.S. Non-Prov. patent application Ser. No. 13/740,843 filed Jan.14, 2013, which issued as U.S. Pat. No. 8,647,886 on Feb. 11, 2014, andU.S. Provisional Application for Patent Ser. No. 61/586,502 filed Jan.13, 2012, the entire contents of which are incorporated herein byreference.

BACKGROUND

The subject technology generally relates to an apparatus, method, systemfor the determination of the aggregation rate of red blood cells. Morespecifically, the subject technology concerns a method, system, and therelative apparatus used to determine the aggregation rate of red bloodcells, and other parameters related to these, such as viscosity,deformability, elasticity, density, in the field of in vitro medicalanalyses, using optical systems after or during inducted forces for redblood cell disruption and redistribution generated by ultrasound waves.

The state of the art for the determination of a test value correspondingto blood subsidence from an aggregogram or syllectogram of a bloodsample is ascertained by reference to the article “Syllectometry, a newmethod for studying rouleaux formation of red blood cells” by Zijlstrapublished in 1963.

Aggregation is the first of three phases describing the sedimentationrate that is composed by: 1) Aggregation 2) Precipitation and 3)Packing. Erythrocyte Sedimentation Rate, which Westergren method isconsidered the gold standard method, is extensively used as a screeningtest for the determination of inflammatory status of a patient.

In the sedimentation phenomenon, aggregation is the first and thefastest among the three phases, which lasts less than two minutes, wherered blood cells (RBC) forming chains (face to face aggregates) termed“Ruloux”. This phase is reversible by mixing action, due, for example,with the repeated inversion of the test tube containing the sample.Rulouxformation causes are still not completely clear; the mostimportant causes are related to proteins dispersed in plasma, such asfibrinogen. However, it is known that aggregation between RBC isstrictly related to infections, inflammatory and connective tissuedisorders.

A second stage aggregation phase, after Ruloux formation, sphericalaggregates are formed between Ruloux with uniform increased mass, thatsediment, after an initial acceleration, at constant speed conformingStokes law. This second phase is called precipitation, and is the phaseevaluated during the Westergren (WG) standard method.

As Stokes law describes that the constant speed is a balance betweengravity force, viscosity and hydrostatic stress. The viscosity in afluid as plasma is heavily affected by thermal effects and can modifysedimentation rate independently of the encountered Ruloux level. Alsolipids dispersed in plasma, in conjunction with lipoproteins, canincrease viscosity and reduce the precipitation phase and the resultingsedimentation rate measure.

Syllectometry is a measuring method that is commonly used to determinethe red blood cell aggregability, which can be related to consequentsedimentation rate. As reference, in syllectometry light is incident toa layer where the sample is exposed to shear stress. Luminous fluxattenuation/increase or backscatter ultrasound wave are used fordetermination of variations in sample density after the abrupt stop ofdriving mechanism. The subsequent time-dependent plot is calledsyllectogram.

Therefore, there remains a need in the prior art for an apparatus,method, system for the determination of the aggregation rate of redblood cells which does not require a stopped flow technique foraggregation kinetic detection.

BRIEF SUMMARY

The subject technology preserves the advantages of prior apparatus,methods, and systems for the determination of the aggregation rate ofred blood cells. In addition, it provides new advantages not found incurrently available apparatus, methods, and systems for thedetermination of the aggregation rate of red blood cells and overcomesmany disadvantages of such currently available systems.

The subject technology generally relates to an apparatus, method, systemfor the determination of the aggregation rate of red blood cells. Morespecifically, the subject technology concerns a method, system, and therelative apparatus used to determine the aggregation rate of red bloodcells, and other parameters related to these, such as viscosity,deformability, elasticity, density, in the field of in vitro medicalanalyses, using optical systems after or during inducted forces for redblood cell disruption and redistribution generated by ultrasound waves.

The subject technology provides a method and the relative reusableapparatus for the determination of aggregation rate index, andsubsequent erythrocytes sedimentation rate for whole blood samples. Thesubject technology reduces the complexity of the pumping systemsremoving the need of the stopped flow condition. The subject technologyprovides other rheological parameters such as viscosity, deformability,elasticity, density. The subject technology provides a method and therelative apparatus for reducing the sample mixing time needed for thedisruption of the aggregates RBC chains, using an alternative methodprior and during the rheological behavior detection. The subjecttechnology reduces the amount of sample volume needed for avoidingcontamination by residuals of previous sample by applying an enhancedwashing system.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features which are characteristic of the subject technologyare set forth in the appended claims. However, the subject technology'spreferred embodiments, together with further objects and attendantadvantages, will be best understood by reference to the followingdetailed description taken in connection with the accompanying drawingin which:

FIG. 1 is a schematic view of an embodiment of the apparatus, method,and system for the determination of the aggregation rate of red bloodcells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the subject technology of FIG. 1, the subjecttechnology generally relates to an apparatus, methods, and systems forthe determination of the aggregation rate of red blood cells. Morespecifically, the subject technology performs a method used to determinethe aggregation rate of red blood cells, and other parameters related tothese, such as viscosity, deformability, elasticity, density, in thefield of in vitro medical analyses, using optical systems after orduring inducted forces for red blood cell disruption and redistributiongenerated by ultrasound waves.

The subject technology provides a method and the relative reusableapparatus for the determination of aggregation rate index, andsubsequent erythrocytes sedimentation rate for whole blood samples. Thesubject technology reduces the complexity of the pumping systemsremoving the need of the stopped flow condition. The subject technologyprovides other rheological parameters such as viscosity, deformability,elasticity, density. The subject technology provides a method and therelative apparatus for reducing the sample mixing time needed for thedisruption of the aggregates RBC chains, using an alternative methodprior and during the rheological behavior detection. The subjecttechnology reduces the amount of sample volume needed for avoidcontamination by residuals of previous sample applying an enhancedwashing system.

In one embodiment, the apparatus 10 for the determination of RBCaggregation, and their subsequent sedimentation rate, according to thesubject technology comprises a reading cell container 16 where thesample is introduced. The apparatus 10 provides this reading cellcontainer 16 equipped with two parallel optical windows for allowinglight radiation to pass through the sample therein introduced or readingthe backscatter of the incident light. The apparatus 10 comprises acollimated light source composed in such way that light passes throughthe windows of the container mentioned above, and can be reflected. Onthe opposite side of the light source 17, there is an optical detector18 for the evaluation of the light attenuated by the sample. The opticaldetector 18 could be positioned on the same side of the light source 17for the detection of light scattering. The reading cell container 16 isequipped with electromechanical actuators 110,111 able to vibrate thesample herein introduced, disrupting the aggregates naturally present inthe blood sample, and evenly distributing the erythrocytes within theentire volume of sample. The apparatus has a temperature control system114, 115 for the sample container to standardize the reactionenvironment.

The apparatus 10 comprises an electronic control device 112 able toacquire the optical variance detected by the optical detector, drive theelectromechanical actuators 110,111 and acquire the containertemperature values. This electronic control device 112 is also able toconvert the detected time dependent light variation into an aggregationindex and the subsequent erythrocyte sedimentation rate, providing theresult of the evaluated phenomenon in the way of a numerical resultcomparable to the commonly used parameters used in a clinicallaboratory.

According to another embodiment of the subject technology, the apparatusor system 10 is comprised of a mixer device 11 for a low homogenizationof the sample inside a collection tube 12. The homogenization can beachieved by a Vortex like mixer or by the radial or axial rotation ofthe sample tube.

After homogenization, the sample is then withdrawn by a needle 13 andaspirated by a pump device 14 through a hydraulic circuit 15. Thehydraulic circuit 15 connects the aspiration needle 13 to the readingcell container 16 to allow their filling by the sample, guaranteed bythe optical sensor composed by the emitter 17 and an optical receiver 18and a secondary optical flow sensor 19 controlled by an electroniccontrol device 112.

The light emitter source 17 is composed, in one embodiment, with a LightEmitting Diode (LED), and can be substituted, for example, by a lasersource or an incandescent lamp. The optical receiver 18, in thisembodiment, may include a CCD sensor for two dimensionalcharacterization of the reaction. This sensor can be substituted with asingle receiver element such as photodiode, photomulfiplier etc.

After the complete or desired filling of the reading cell 16 the pumpdevice 14 is stopped by the electronic control device 112, and thesample is processed by the electromechanical devices 110, 111, forexample composed by piezoceramics, activated to a predetermined power bythe control device 112, to disrupt aggregates and evenly re-suspend theRBC on the sample volume. A prerequisite for an aggregation kineticdetection is a complete disruption of the aggregates, normally formed ina steady state of the sample. This disruption can be achieved by anintensive mixing phase before and during the transportation of thesample in the reading cell or detection.

As an alternative to a predetermined power, the piezoceramic power isinitially ramped up to a level where cell emulsification is detectedthrough the optical reading. This process is stopped and a duplicatesample is introduced. The power applied can be optimized at a fractionof the emulsification power level which results in maximum dispersion,without cell damage.

During this phase the control device 112 acquires the signal detected bythe optical receiver 18 and stops the electromechanical devices 110, 111or actuators when the light variation detected by the receiver 18 stopsdecreasing, indicating the complete disruption of the aggregate presentinto the sample. This recorded plot expresses the disruption rate of theRBC and is post evaluated by the system.

In one embodiment, the shape of the reading cell container 16 wallscomprises sound lenses for focusing the wave pressure shear to emphasizethe shear inducted to the sample.

After the electromechanical devices 110,111 stop, the signal detected bythe receiver 18 is still recorded by the control device 112 for apredetermined amount of time as a plot of kinetic aggregation.

After the end of the acquisition the sample is evacuated from thereading cell 16 by the pump device 14 to a waste reservoir 113. Duringthe evacuation, the electromechanical devices 110, 111 are activatedwith a high power to remove proteins bonded to the walls of the readingcell container 16. An evacuation of the reading chamber avoids thepollution of the sample currently under measure by the residual of theprevious measured sample with washing and does not require a large flowamount of sample currently under measure for removal the residuals ofthe previous measured sample. After the evacuation, the system is readyfor a new sample and analysis.

The reading cell container 16 is also maintained to a controlledtemperature by the thermoelectric device 114 and the temperature isacquired by the control device 112 through the temperature sensor 115for providing standardized conditions of reaction.

During the dispersion phase induced by the electromechanical devices110, 111, the resultant signal is evaluated to extract the meanviscosity value of the sample plasma by considering the time needed bythe sample to completely re-suspend. After a complete re-suspension ofthe sample, a burst of ultrasound waves is induced to the sample forevaluating the red blood cell deformability. This deformability isconsidered as the time needed by the media to absorb the wave shearimpressed, also decay after the wave share absorption is evaluated infunction of time as index of the mean shape recovery ability.

It should be appreciated that the system, method, and apparatus mayinclude one or more components or steps listed above in a variety ofconfigurations depending upon desired performance or requirements.

It would be appreciated by those skilled in the art that various changesand modifications can be made to the illustrated embodiments withoutdeparting from the spirit of the subject technology. All suchmodifications and changes are intended to be within the scope of thesubject technology.

1-14. (canceled)
 15. An apparatus for determining the aggregation rateof red blood cells, comprising: an optical receiver positioned to detectlight from a blood sample portion comprising red blood cells that haveaggregated; and a main controller coupled to the optical receiver forrecording an aggregation rate of the red blood cells of the blood sampleportion for a predetermined time based upon detected light variation.16. The apparatus of claim 15, further comprising: a hydraulic circuitfor providing the blood sample portion; a reading cell containerconnected to the hydraulic circuit for receiving the blood sampleportion; and a light emitter source to pass light into the blood sampleportion.
 17. The apparatus of claim 16, further comprising: a disruptionmechanism connected to the reading cell container for disruption of thered blood cells within the blood sample portion to assist in recordingthe disruption rate, wherein the main controller activates thedisruption mechanism for the disruption of the red blood cells withinthe blood sample portion until light detected indicates the disruptionof aggregate within the blood sample portion.
 18. The apparatus of claim16, further comprising: a fluid reservoir connected to the reading cellcontainer for receipt of an evacuated blood sample portion from thereading cell container.
 19. The apparatus of claim 18, wherein theapparatus comprises an evacuation mechanism to evacuate the evacuatedblood sample portion from the reading cell container, wherein theevacuation mechanism is configured to provide ultrasound stress to thereading cell container.
 20. The apparatus of claim 16, wherein thereading cell container is configured for the optical detection ofaggregation reaction.
 21. The apparatus of claim 15, wherein theapparatus determines a disruption index of the red blood cells asrheological parameters usable for pathologic detection purposes.
 22. Theapparatus of claim 15, wherein the apparatus determines a mean red bloodcells shape recovery ability.
 23. The apparatus of claim 15, wherein theapparatus determines the plasma viscosity.
 24. A method of measuring thedisruption rate and the aggregation rate of a sample of red blood cells,the method comprising the steps of: obtaining a blood sample portioncomprising aggregated red blood cells; delivering the blood sampleportion into a reading cell container; using a disruption mechanism todisrupt the aggregates in the blood sample portion within the readingcell container to distribute and re-suspend the red blood cells; passinglight from a light emitter source into the blood sample portion withinthe reading cell container; receiving and detecting light emitted fromthe reading cell container at an optical receiver; recording, at a maincontroller, a disruption rate of the red blood cell aggregates withinthe blood sample portion based upon the light variation; and recordingan aggregation rate of the blood sample portion for a predetermined timebased upon detected light variation.
 25. The method of claim 24, whereinthe step of using the disruption mechanism includes applying an optimumemulsification power level of electricity to a piezoceramic materialdetermined by initially ramping up electricity applied to thepiezoceramic material to a level where cell emulsification of a testsample is detected through the reading cell by the optical receiver. 26.The method of claim 24, further comprising the step of discontinuing thedisruption of the blood sample portion within the reading cell until thevariation in light detected indicates the disruption of the red bloodcell aggregates within the blood sample portion.
 27. An apparatus fordetermining an aggregation rate of red blood cells comprising: a readingcell container configured to receive a blood sample portion comprisingaggregated red blood cells; an optical receiver positioned to detectlight from the blood sample portion while the blood sample portion is inthe reading cell container; a disruption mechanism connected to thereading cell container to disrupt the aggregated red blood cells withinthe blood sample portion while the blood sample portion is in thereading cell container; and a main controller configured to record anaggregation rate of the red blood cells of the blood sample portionbased upon detected light variation.
 28. The apparatus of claim 27,further comprising: a hydraulic circuit for transporting a blood sampleportion comprising red blood cells; and a light emitter source to passlight through the blood sample portion while the blood sample portion isin the reading cell container.
 29. The apparatus of claim 27, furthercomprising: a fluid reservoir connected to the reading cell containerfor receipt of an evacuated blood sample portion from the reading cellcontainer; and an evacuation mechanism to evacuate the evacuated bloodsample portion from the reading cell container, wherein the evacuationmechanism is configured to provide ultrasound stress to the reading cellcontainer.
 30. The apparatus of claim 27, wherein the main controllerdetermines the aggregation rate based upon for a predetermined time ofdisruption.
 31. The apparatus of claim 27, wherein the main controllerdetermines the aggregation rate based upon data collected from a startof disruption until light detected stops decreasing.
 32. The apparatusof claim 27, wherein the detected light is backscattered.
 33. Theapparatus of claim 27, wherein the detected light passes through theblood sample and is attenuated.
 34. The apparatus of claim 27, whereinthe detected light includes first light that is backscattered and secondlight that has passed through the blood sample.